With an ever increasing world population there is continuing expansion in the need for high quality protein to meet human nutrient requirements. However, because of limitations in agricultural land, there will be an increasing requirement for the biosynthesis of food proteins using fermentation processes employing various microorganisms. Thus, various bacteria and yeast which can grow on inexpensive substrates can produce copious amounts of high quality proteins. This protein has great potential for use in foods. However, for the protein to be effectively used in food manufacturing and food processing it is essential that it be initially extracted and refined. One of the major problems limiting the greater use of proteins from microbes, particularly for human consumption and also for use in pet foods (particularly for monogastric animals), is the high nucleic acid content of these protein preparations. The intake of relatively high quantities of nucleic acid is undesirable because it can cause uricemia, gout and kidney stone formation. For humans, the Protein Advisory Group of the United Nations has recommended that the amount of nucleic acid ingested per day should not exceed 2 grams, and in fact, it should be much less. This means that the nucleic acid content of microbial proteins intended for food use should be less than 5% if the microbial protein supplies 50% of the dietary protein. However, proteins prepared by conventional methods from microbial sources contain from 10-27% grams/100 grams dry protein. Therefore, a practical method is needed for the reduction of the nucleic acid in proteins from microbial sources.
Furthermore, with the rapid development of recombinant DNA techniques and genetic engineering, microbes will be increasingly used for the production of materials for agricultural, biomedical and industrial uses. Thus, genetic engineering is and will be used to an increasing extent for the production of hormones; for the production of enzymes for use in biomedical and industrial applications such as in the food industry for the production of high quality proteins. However, a major difficulty in this regard is the effective separation of these proteinaceous materials from the nucleic acid materials present in the microbial cells. Because of the tremendous potential in these areas there is an urgent need for the development of a practical method for the isolation of these proteinaceous materials with a minimum of nucleic acid contamination.
In addition, the demand for nucleic acids for biomedical research (recombinant DNA) and uses in commercial products (shampoos, cosmetics) is increasing. However, the present methods for preparing large lots of these is expensive and time-consuming. The procedures described herein affords a practical method for the simultaneous separation of protein and nucleic acid for research and commercial applications.
With this background information it is obvious that a practical method, compatible with existing processing technology, which would facilitate the isolation of nucleic acids and proteinaceous material devoid of nucleic acid needs to be developed. The invention described herein provides such a method.
Several laboratory scale methods have been proposed for the reduction of nucleic acid levels in proteins isolated from microbial cells. These methods involve the chemical and enzymatic treatments of the protein-nucleic acid complex to degrade the nucleic acids and to facilitate their separation based on electrostatic charge repulsion. However, these methods entail extra steps for isolation; they are costly; they may involve the formation of potentially toxic compounds such as lysinoalanine, or they may result in the production of chemically derived proteins which are of questionable value for human nutrition.
The nucleic acids (mostly ribosomal ribonucleic acids) in microbial cells exist in the form of tightly bound nucleoprotein complexes. Consideration of the fundamental mechanism of these interactions strongly suggests that hydrophobic forces are the principle forces responsible for the formation of these protein nucleic acid complexes. Thus, destabilization of the hydrophobic interactions between proteins and nucleic acids should facilitate the separation and preparation of microbial proteins and nucleic acids. Because the principal driving force for hydrophobic associations between molecules critically depends upon the structural state of surrounding water molecules, manipulating the structure of water may cause the destabilization of hydrophobic interactions, thereby facilitating separation of the proteins and nucleic acids. Such changes in the structure of water can be induced by chaotropic salts.